This career development award will facilitate the training needed for the principle investigator (PI) to transition to a tenure-track faculty positio and establish an independent research program focused on using developmental biology principles to generate biomimetic constructs for regenerative craniofacial therapy. The ultimate goal of this research is to engineer customized, multi-tissue structures to replace dental tissues. Tissue-engineered constructs could restore function and aesthetics to patients suffering from trauma, congenital defects, and disease to the craniofacial region. However, the craniofacial region comprises complex, multi-tissue structures that are challenging to recreate using traditional tissue engineering methods due to difficulties in directing correct spatial organizatio and producing proper interfaces. During natural tissue development, cell migration and the expression of morphogen gradients are two critical components of tissue patterning; understanding and controlling these processes is key for improving tissue engineering design. The PI will acquire training to study and manipulate cell migration and the expression of morphogen gradients to investigate mechanisms facilitating the formation of organized multi-tissue structures in engineered tissues and to customize tissue patterning. The PI has previously shown that forming novel three-dimensional, scaffold-free tissue-engineered constructs (SFC) from dental pulp cells (DPC) results in spatially organized dentin-pulp complex-like structures containing a pulp-like core enclosed within a dentin-like periphery. In Aim 1, the PI will receive training in advanced imaging techniques to track cell migration within 3D tissues. The hypothesis that in SFCs generated from the total population of dental pulp cells, the CD146+ stem/progenitor cells migrate to the periphery to produce the outer dentin-like tissue will be tested. In Aim 2, the PI will acquire training in microfluidic devices to generate molecular gradients across SFCs to alter tissue patterning. Gradients of calcium and transforming growth factor ?1, known factors involved in odontogenic differentiation, will be generated across the SFCs to test the hypothesis that a dentin-like structure will form in in the highest concentrations of the chemical gradients and transition to a pulp-like tissue. As an independent investigator the PI will use the training received in Aim 1 to track stem/progenitor cell migration in SFCs in response to the molecular gradients generated by the microfluidic devices. In Aim 3, as an independent investigator, the PI will generate a cementum-like tissue layer on the outer surface of the dentin-pulp complex-like SFC. SFCs generated by DPCs will be wrapped with a periodontal ligament stem/progenitor cell (PDLC) sheet to test the hypothesis that PDLC will differentiate towards a cementoblast phenotype when spatially positioned on an outer surface of a dentin-pulp complex-like structure. Additionally, using the training received in Aim 2, microfluidic devices will be used to generate gradients of bone morphogenic protein 2 or insulin-like growth factor 1, known inducers of cementoblast differentiation, across the SFCs to alter cementum patterning.